The present invention is in the field of enzyme proteins that are related to the NADPH oxidase subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Many human enzymes serve as targets for the action of pharmaceutically active compounds. Several classes of human enzymes that serve as such targets include helicase, steroid esterase and sulfatase, convertase, synthase, dehydrogenase, monoxygenase, transferase, kinase, glutanase, decarboxylase, isomerase and reductase. It is therefore important in developing new pharmaceutical compounds to identify target enzyme proteins that can be put into high-throughput screening formats. The present invention advances the state of the art by providing novel human drug target enzymes related to the NADPH oxidase subfamily.
Neutrophil NADPH oxidase is a multicomponent enzyme that is activated to generate superoxide anion and is defective in the cells of patients with chronic granulomatous disease. It requires both membrane and cytosolic components, the latter including 47- and 67-kDa proteins recognized by the polyclonal antiserum B-1. Immunoscreening of an induced HL-60 lambda ZAP cDNA library yielded seven cross-hybridizing cDNAs encoding the 47-kDa component. Fusion proteins of 22-50 kDa were recognized by B-1. Antiserum against a fusion protein recognized a 47-kDa protein in normal neutrophils but not in those from patients with autosomal chronic granulomatous disease who lack the 47-kDa cytosolic oxidase component.
The phagocyte NADPH oxidase is a complex enzyme system that plays an important role in host defense. After stimulation with opsonized microorganisms or other activating agents, the oxygen consumption of these cells increases dramatically (respiratory burst) and they release a large amount of superoxide. Superoxide is then converted to more potent reactive oxygen species such as hydrogen peroxide, hydroxyl radical, and hypohalous acids, which are used by phagocytes to control microbial infections. The importance of this defense mechanism is made evident by a rare inherited syndrome, chronic granulomatous disease (CGD) in which phagocytes fail to generate superoxide, rendering the patients highly susceptible to life-threatening microbial infections.
The present invention has a substantial similarity to p47phox. The cDNA of this enzyme was originally isolated for the 47-kilodalton (kDa) subunit of the NADPH oxidase system, whose absence is responsible for the most common form of autosomally inherited chronic granulomatous disease (CGD). It encodes a 44.7-kDa polypeptide, which contains two src homology (SH3) domains and several possible sites for phosphorylation by protein kinase C. An antiserum raised to the predicted C terminus of the protein detects a polypeptide with an apparent molecular mass of 47 kDa in normal neutrophil granulocytes but not in those from patients with autosomal CGD. The antibody has been used to show that the protein associates with the vacuolar membrane and is phosphorylated in response to phorbol ester treatment. Analysis of a number of tissue types and cell lines shows that expression of the gene is confined to phagocytic cells and B lymphocytes. It is suggested that patients with CGD may also have a defect in lymphocyte function. p47 protein and mRNA levels increase during retinoic acid-induced neutrophil differentiation of HL60 cells. The gene is a primary target for regulation by retinoic acid. For a review related to p47 phox, see Rodaway et al., Mol Cell Biol October 1990;10(10):5388-96; Volpp et al., Proc Natl Acad Sci USA September 1989;86(18):7195-9 Erratum in: Proc Natl Acad Sci USA December 1989;86(23):9563.
Enzyme proteins, particularly members of the NADPH oxidase subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of enzyme proteins. The present invention advances the state of the art by providing previously unidentified human enzyme proteins, and the polynucleotides encoding them, that have homology to members of the NADPH oxidase subfamily. These novel compositions are useful in the diagnosis, prevention and treatment of biological processes associated with human diseases.
The present invention is based in part on the identification of amino acid sequences of human enzyme peptides and proteins that are related to the NADPH oxidase subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate enzyme activity in cells and tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, B cells from Burkitt lymphoma, primary B-cells from tonsils and leukocyte.